Over the past two decades, the in vitro amplification of specific nucleic acids has become an essential tool for molecular biologists.
More recently, multiplexed amplification, in which a plurality of nucleic acid sequences are amplified in a single reaction, Chamberlain et al., Nucl. Acid Research 16(23):11141-1156 (1988); U.S. Pat. No. 5,582,989, has become increasingly important. For example, multiplexed amplification, particularly multiplexed polymerase chain reaction (PCR), has been used to provide genetic fingerprints of infectious disease organisms. Other applications, such as multiplex SNP genotyping and variation scanning (for example, by mismatch repair detection), also greatly benefit from PCR multiplexing.
In its original implementation, multiplex PCR reactions include a specific primer pair for each locus to be amplified. These approaches have been plagued with problems, however, including uneven or failed amplification of some templates (especially those having GC rich-sequences), preferential amplification of other templates, poor sensitivity and specificity, poor reproducibility, and the generation of spurious amplification products (Henegariu et al., BioTechniques 23(3): 504-511 (1997); Markoulatos et al., J. Clin. Lab. Anal. 16: 47-51 (2002)).
Various modifications to the original approach have been developed in efforts to minimize these problems.
Among these modifications are changes to the reaction conditions, including adjustment of primer concentrations, MgCl2 and dNTP concentrations, changes in PCR buffer concentrations, balance between MgCl2 and dNTP concentrations, amounts of template DNA and Taq DNA polymerase, extension and annealing time and temperature, and the addition of adjuvants (Henegariu et al., BioTechniques 23(3): 504-511 (1997); Markoulatos et al., J. Clin. Lab. Anal. 16: 47-51 (2002)). Other strategies used include subcycling temperatures between high and low temperatures below the denaturation temperature, used during the annealing and elongation steps (U.S. Pat. No. 6,355,422), and the use of one sequence-specific primer and one common primer (Broude et al., Proc. Natl. Acad. Sci. USA 98, 206-211 (2001)).
The intractability of GC-rich sequences to multiplex PCR has also been addressed by a method in which addition of betaine and dimethylsulfoxide (DMSO) to the PCR reaction mix is said to allow more uniform amplification from a heterogeneous population of DNA molecules, many of which were GC-rich (Baskaran et al., Genome Research 6: 633-638 (1996)).
Yet other approaches alter the primers.
In one such effort, chimeric oligonucleotides are used as primers: the oligonucleotides include a 3′ domain that is complementary to template, conferring template specificity, and a 5′ domain that is noncomplementary to template; the 5′ domain includes a sequence used to prime extension in rounds of PCR amplification subsequent to the first.
In this latter scheme, however, the cycles of amplification following the first amplify whatever product is generated in the first cycle, whether correct or erroneous. Thus, while the technique allows for more uniform amplification, it does not address the problem of spurious products.
In an analogous approach designed to clone the shared components in two complex samples, Brookes et al., Human Molec. Genetics 3(11):2011-2017 (1994), ligate primers to template ends generated by restriction fragment digestion.
None of the above-mentioned approaches, however, fully solves the problems associated with multiplex PCR.
Thus, there is a continuing need in the art for a method that allows the specific and uniform amplification of multiple nucleic acid sequences in a single reaction, without the generation of spurious products.